Arthropod-borne viruses (arboviruses) constitute a persistent and worldwide challenge to public health. Maintenance of mosquito-borne viruses in nature requires a biological transmission cycle that involves alternating virus replication in a susceptible vertebrate and mosquito host. For arboviruses in nature, it is imperative that very little fitness cost be associated with infection of the mosquito. We recently demonstrated the importance of RNA interference (RNAi) in the mechanism by which alphaviruses establish a persistent, nonpathogenic infection in the mosquito vector, showing that in the absence of RNAi-based modulation, mosquitoes do not survive arboviral infection. However, very little direct experimentation has been done on mosquito RNAi genes. We have described the generation and validation of a transgenic strain of Aedes aegypti that "senses" the status of the RNAi pathway, through which we have shown that the genes Dcr-2 and Ago-2, but not Ago-3, are critical for RNAi in Ae. aegypti. We hypothesize that genetic variability in the RNAi pathway directly affects the ability of mosquitoes to become infected by, and transmit, arboviruses. In specific aim 1, we will use our transgenic "sensor strain" to determine the involvement of mosquito genes in the regulation and execution of RNAi. In specific aim 2, we will compare primary nucleotide sequences of various mosquito genes within the genus Aedes, and determine whether any evolutionary selective pressures are acting on genes involved in RNAi. In specific aim 3, we will determine the role of genetic variability in RNAi genes on the vector competence of Aedes mosquitoes for medically important arboviruses. A detailed understanding of mosquito RNAi genes, and the role played by genetic variation in those genes on virus transmission, will facilitate better evaluations of the feasibility of RNAi-based genetic control strategies, and allow us to more accurately determine the risks of new and emerging arboviruses spreading to new areas, particularly within the U.S. PUBLIC HEALTH RELEVANCE: Arthropod-borne virus (arbovirus) diseases such as yellow fever, dengue fever, and chikungunya fever remain a significant burden on global public health. This proposal deals directly with the viral pathogens that cause those diseases. We aim to characterize novel components of the mosquitoes'innate immune system which acts as a general constraint to the accumulation of virus in the mosquito. This knowledge is critical to understanding and predicting the emergence of new arboviral epidemics, as well as to understanding how arboviruses are maintained in nature, and may ultimately form the basis of RNAi-based transgenic and field diagnostic approaches for disease control.